Shared-autosampler parallel LC-MS/MS platform for high-throughput multi-analyte quantification in complex matrices

Modern analytical tasks increasingly require the simultaneous quantification of large numbers of chemically diverse analytes in complex matrices, which remains challenging for a single LC-MS method. Here, we developed a shared-autosampler parallel LC-MS/MS strategy that enables two fully independent chromatographic methods to be executed sequentially within a single analytical cycle while generating one unified chromatogram-mass spectrum. The system employed two LC pump modules and two columns to form two independent LC pathways, which shared one autosampler and one MS detector for selective MRM acquisition and subsequent signal integration. Integration was performed exclusively at the MS detection and data-processing level, without inter-dimensional coupling or analyte transfer between columns. This avoided the mobile-phase compatibility and dead-volume limitations associated with 2D-LC, enabled modular recombination of chromatographic and MS conditions, and improved analytical throughput without additional MS detector or autosampler hardware. The performance of the strategy was demonstrated in two applications. An acidic/basic dual-method configuration enabled quantitative analysis of 394 emerging contaminants from 14 chemical classes in human serum within 28 min, exhibiting good linearity, limits of quantification not exceeding 0.20 ng/mL for >90 % of analytes, and peak-area RSDs generally below 10 %. A HILIC/RP parallel-column combination achieved simultaneous determination of 27 hypoglycemic agents spanning a log P range of -6.8 to 5.9 in a single 20 min run, reducing analysis time by >50 % compared with standard methods, with good precision and recoveries in spiked food samples. Overall, the shared-autosampler parallel LC-MS/MS strategy provided a flexible and efficient platform for high-throughput multi-analyte quantification in complex matrices.